Applied and Environmental Microbiology最新文献

CRISPR-Cas systems are transforming precision medicine with engineered probiotics as next-generation diagnostics and therapeutics. To promote human health and treat disease, engineering probiotic bacteria demands maximal versatility to enable non-natural functionalities while minimizing undesired genomic interferences. Here, we present a streamlined prime editing approach tailored for probiotic Escherichia coli Nissle 1917 utilizing only essential genetic modules, including Cas9 nickase from Streptococcus pyogenes, a codon-optimized reverse transcriptase, and a prime editing guide RNA, and an optimized workflow with longer induction. As a result, we achieved all types of prime editing in every individual round of experiments with efficiencies of 25.0%, 52.0%, and 66.7% for DNA deletion, insertion, and substitution, respectively. A comprehensive evaluation of off-target effects revealed a significant reduction in unintended mutations, particularly in comparison to two different base editing methods. Leveraging the prime editing system, we inserted a unique DNA sequence to barcode the edited strain and established an antibiotic-resistance-gene-free platform to enable non-natural functionalities. Our prime editing strategy presents a CRISPR-Cas system that can be readily implemented in any laboratories with the basic CRISPR setups, paving the way for future innovations in engineered probiotics.IMPORTANCEOne ultimate goal of gene editing is to introduce designed DNA variations at specific loci in living organisms with minimal unintended interferences in the genome. Achieving this goal is especially critical for creating engineered probiotics as living diagnostics and therapeutics to promote human health and treat diseases. In this endeavor, we report a customized prime editing system for precision engineering of probiotic Escherichia coli Nissle 1917. With such a system, we developed a barcoding system for tracking engineered strains, and we built an antibiotic-resistance-gene-free platform to enable non-natural functionalities. We provide not only a powerful gene editing approach for probiotic bacteria but also new insights into the advancement of innovative CRISPR-Cas systems.

The airborne transmission of infectious diseases and bioaerosol-induced cross-contamination pose significant challenges in the food, dairy, and pharma industries. This study evaluated the effectiveness of 279 nm UV-C LED irradiation for decontaminating bioaerosols, specifically containing microorganisms such as Escherichia coli (C3040- Kanamycin resistant), Salmonella Enteritidis (ATCC 4931), and Pseudomonas fragi (ATCC 4973), on food contact surfaces. Borosilicate glass, silicon rubber, and stainless steel (316L) surfaces were selected for experimentation for their usage in the food industry. A 50 µL cell suspension was aerosolized at 25 psi pressure using a 4-jet BLAM Nebulizer within a customized glass chamber and then deposited onto the surface of the coupons. The serial dilution approach was used for the microbial enumeration, followed by duplicate plating. With a low Root Mean Square Error (RMSE) and high R² values, the biphasic kinetic model for UV-C inactivation curves of all three pathogens demonstrated the excellent goodness of fit parameters. At a UV-C dose of 6 mJ cm^-2, glass surfaces showed the maximum microbial inactivation (i.e., 2.80, 3.81, and 3.56 log CFU/mL for E. coli, Salmonella, and P. fragi, respectively). Stainless steel and silicon rubber surfaces showed significant microbial inactivation, but log₁₀ reductions observed were consistently lower than glass surface. Our research indicates that UV-C LEDs (279 nm) can effectively disinfect bioaerosols on food contact surfaces.IMPORTANCEFood safety is a major public health concern, with contaminated food causing serious illnesses. UV-C light, used for germicidal action, is effective in disinfecting surfaces and is not subject to the same strict legal restrictions as chemical disinfectants, simplifying compliance with food safety regulations. In this study, we evaluated the efficacy of UV-C (279 nm) LED systems for inactivation of surface-deposited bioaerosols of kanamycin-resistant Escherichia coli (C3040), Salmonella Enteritidis (ATCC 4931), and Pseudomonas fragi (ATCC 4973). The research outcomes can be used to develop UV-based surface disinfection systems to minimize the risk of foodborne illnesses and enhance safety in high-traffic food preparation areas.

Coagulase-negative staphylococci (CoNS) comprise about 50 species, some of which cause septicemia in preterm neonates. CoNS establish early on the skin and in the oral and gut microbiota, from where they may spread to the bloodstream. The colonization pattern preceding septicemia is not well-defined. Forty-two extremely preterm neonates (≤28 + 0 gestational weeks) were followed from birth to 2 months with regular sampling and culturing of the skin and oral and gut microbiota. Blood samples were drawn upon clinical suspicion of septicemia and cultured. CoNS species were identified using matrix-assisted laser-desorption ionization time of flight mass spectrometry (MALDI-TOF). Random amplified polymorphic DNA was used for strain typing, and strains were characterized regarding biofilm production and virulence gene carriage. CoNS blood isolates underwent whole genome sequencing. Staphylococcus epidermidis represented 72% of the CoNS isolates on skin or mucous membranes, followed by Staphylococcus capitis (13%) and Staphylococcus haemolyticus (7%). CoNS septicemia was diagnosed in nine infants, yielding 11 septicemia isolates: seven S. capitis and four S. epidermidis, of which nine were further analyzed. The S. capitis septicemia isolates belonged to the NRCS-A clone. Two-thirds of the septicemia strains were traced back to the commensal microbiota. Colonization of the oral cavity by S. capitis was significantly associated with CoNS septicemia development, although the blood-borne S. capitis strains were more commonly found on the skin than in the mouth prior to invasion. Biofilm production was not associated with septicemia. Our results implicate CoNS colonization as a step that precedes septicemia in preterm neonates. Early colonization of the oral cavity by S. capitis may represent a particular risk.

Importance: Septicemia is a major cause of morbidity in preterm infants. Coagulase-negative staphylococci (CoNS) can colonize skin, oral cavity, and intestines and are a common cause of septicemia in this group. The relation between CoNS colonization pattern at the species and strain level and septicemia has scarcely been studied. We mapped colonization of the skin, oral cavity, and intestines by CoNS species in extremely preterm infants and speciated and strain-typed the skin, mucosal, and blood isolates. Two-thirds of the CoNS septicemia blood strains, including a majority of S. capitis strains belonging to the NRCS-A clone, were tracked to the commensal microbiota. We demonstrated that CoNS species differ in their colonization patterns, whereby S. capitis was primarily a skin colonizer. However, its colonization of the oral cavity was enhanced among infants developing septicemia. Our study provides a starting point for further explorations of the relationship between CoNS colonization and septicemia in preterm infants.

To achieve rapid and simultaneous detection of NoV GI, NoV GII, and HAV, a quadruple real-time fluorescence quantitative PCR (RT-qPCR) assay was developed using MS2 bacteriophage as a process control virus. The quadruple RT-qPCR assay effectively detected NoV GI, NoV GII, HAV, and MS2 RNA with detection limits of 10² copies/μL, 10³ copies/μL, 10² copies/μL, and 10³ copies/μL, respectively, within 1 hour 50 minutes. The quadruple RT-qPCR assay could specifically detect NoV GI, NoV GII, HAV, and MS2 without cross-reactions with other common pathogens, demonstrating good reproducibility with intra-assay and inter-assay coefficients of variation all below 2.11%. In this study, 337 bivalve shellfish samples collected from various regions of Hebei Province were pretreated using the proteinase K-PEG 8000 precipitation-chloroform method, and viral nucleic acids were enriched and extracted from a volume of viral solution that was doubled. The developed quadruple RT-qPCR assay was used to detect NoV GI, NoV GII, and HAV in bivalve shellfish samples, and the positive rates were 19.88% (67/337), 20.47% (69/337), and 4.75% (16/337), respectively. In addition, mixed infections of NoV GI and NoV GII (10.68%, 36/337) and NoV GI and HAV (0.89%, 3/337) were observed. In all, 200 bivalve shellfish samples were randomly selected for the assay, and it was found that the total, positive, negative coincidence rates, and Kappa values of the quadruple RT-qPCR assay were 98.3%, 99.1%, 98.2%, and 0.945, respectively, compared with the single RT-qPCR assay. These results show that the developed quadruple RT-qPCR assay has comparable performance to the single RT-qPCR assay.IMPORTANCEFood-borne diseases caused by viral contamination have become a growing concern, and bivalve shellfish is a crucial source of infection, with many outbreaks of non-bacterial acute gastroenteritis associated with raw food or the use of undercooked shellfish such as oysters. As food contamination problems caused by NoV and HAV become more severe, it is important to study and establish a sensitive and efficient assay to simultaneously detect NoV and HAV by applying the MS2 process control virus for the protection of bivalve shellfish food safety and the monitoring of the above food-borne viral contamination. In addition, bivalve shellfish samples contain a large number of PCR inhibitors such as polysaccharides, lipids, and proteins, so optimization of the virus enrichment and extraction method is essential and is expected to provide a research basis for subsequent related experiments.

Postdocs are essential to microbial science and STEM academic workforces but are underpaid and receive little-to-no relocation benefits. PhDs foregoing postdoctoral training for lucrative industry and government jobs exit the academic pipeline, which imperils current scholarship and the future professoriate. Relocation to postdoc jobs is expensive, especially for recent graduates and international scholars, but academia rarely provides support. Solving this short-term liquidity pressure can increase productivity, job satisfaction, and the likelihood they remain in academia.

Ecological processes greatly shape microbial community assembly, but the driving factors remain unclear. Here, we compiled a metagenomic data set of microbial communities from global acid mine drainage (AMD) and explored the ecological features of microbial community linked to stochastic and deterministic processes from the perspective of species niche position, interaction patterns, gene functions, and viral infection. Our results showed that dispersal limitation (DL) (48.5%~93.5%) dominated the assembly of phylogenetic bin in AMD microbial community, followed by homogeneous selection (HoS) (3.1%~39.2%), heterogeneous selection (HeS) (1.4%~22.2%), and drift (DR) (0.2%~2.7%). The dominant process of dispersal limitation was significantly influenced by niche position in temperature (r = -0.518, P = 0.007) and dissolved oxygen (r = 0.471, P = 0.015). Network stability had a significantly negative correlation with the relative importance of dispersal limitation, while it had a positive correlation with selection processes, implying changes in network properties could be mediated by ecological processes. Furthermore, we found that ecological processes were mostly related to the gene functions of energy production and conversion (C), and amino acid transport and metabolism (E). Meanwhile, our results showed that the number of proviruses and viral genes involved in arsenic (As) resistance is negatively associated with the relative importance of ecological drift in phylogenetic bin assembly, implying viral infection might weaken ecological drift. Taken together, these results highlight that ecological processes are associated with ecological features at multiple levels, providing a novel insight into microbial community assembly in extremely acidic environments.

Importance: Unraveling the forces driving community assemblage is a core issue in microbial ecology, but how ecological constraints impose stochasticity and determinism remains unknown. This study presents a comprehensive investigation to uncover the association of ecological processes with species niche position, interaction patterns, microbial metabolisms, and viral infections, which provides novel insights into community assembly in extreme environments.